The established method in many laboratories for quantifying endotoxin - in other words components of the lipopolysaccharides of the outer cell membrane of gram-negative bacteria - is the Limulus amoebocyte lysate (LAL) test. The first in vitro test for the detection of endotoxins and heat-stable cell wall components of gram-negative bacteria and blue-green algae was developed in the 1970s. However, the test is based on a reaction of the blood of the horseshoe crab (Limulus polyphemus). An infection with gram-negative bacteria leads to blood clotting, as the bacterial endotoxin reacts with a gel-forming protein in the blood cells (amoebocytes) of the animals. This test is no longer mandatory in the United States.

Horseshoe crab (Limulus polyphemus).
Graphic: Computer-generated.

There are leading representatives in both the industry and the US Congress who are in favor of synthetic test alternatives. In Congress, Representative Frank Pallone and Senator Cory Booker, both from New Jersey, have supported the adoption of the amendment to Chapter 86. The alternatives are safe, effective and do not incur the environmental costs that LAL does, they say. The pharmaceutical company Eli Lilly has been using an animal-free alternative, recombinant factor C, since 2016.

The increasing global demand for e.g. vaccines, and therefore also for the blood of horseshoe crabs, has long put their populations under severe pressure and also threatened biodiversity. The population of the red knot (Calidris canutus), for example, depends on the health of the horseshoe crabs, on whose eggs it relies for food. Europe must now quickly follow this trend.

The so-called monocyte activation test (MAT) would also be suitable as a replacement method. The test uses an innate human immune response to so-called pyrogens, i.e. bacterial substances such as cell wall fragments, particles of yeasts, fungi or viruses. At its 179th meeting in June 2024, the European Pharmacopoeia Commission adopted 57 revised texts, which resulted in the complete deletion of the test for fever-inducing substances in rabbits (pyrogen test) from the European Pharmacopoeia.
Instead of the rabbit, which had not always reliably indicated the fever reaction in all cases, the MAT or another test must now be used. Unfortunately, they had not negotiated about the LAL with blood from horseshoe crabs.

The MAT has a much broader detection spectrum than the Limulus Amebocyte Lysate (LAL) test, which can only detect lipopolysaccharides of Gram-negative bacteria (endotoxins) to a limited extent.

Sources:
https://www.humanesociety.org/news/horseshoe-crab-recovery-coalition-celebrates-us-pharmacopeia-recognition-synthetic
https://pallone.house.gov/media/press-releases/pallone-urges-swift-use-new-biomedical-guidelines-protect-horseshoe-crabs-and
https://www.researchgate.net/
https://www.invitrojobs.com/index.php/en/news/news-archive/item/6482-tests-for-fever-inducing-substances-rabbit-use-finally-deleted
https://www.invitrojobs.com/index.php/en/news/news-archive/item/5936-monocyte-activation-test-revision-of-the-european-pharmacopoeia
https://www.invitrojobs.com/index.php/en/research-and-methods/working-group-a-portrait/item/1280-working-group-a-portrait-clinical-research-laboratory